Chemically de-acetylated 2 ',7 '-dichlorodihydrofluorescein diacetate as aprobe of respiratory burst activity in mononuclear phagocytes

2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) is a fluorogenic pro be commonly used to detect cellular production of reactive oxygen species ( ROS), for example in the respiratory burst of granulocytes and mononuclear phagocytes. This method depends on the de-acetylation of H(2)DCFDA by cellu lar esterases, to form the oxidant-sensitive compound, 2',7'-dichlorodihydr ofluorescein (H2DCF). Importantly, however, not all cells possess sufficien t esterase activity to produce the H2DCF needed for accurate measurement of ROS. In this study, we used chemically de-acetylated probe (H2DCF) to asse ss the phorbol-ester-triggered respiratory burst of rainbow trout macrophag es, which, like some mammalian mononuclear phagocytes, appear to have low p robe-esterase activity. We compared this approach to the use of intact H(2) DCFDA and the cytochrome c reduction assay. The H2DCF and cytochrome c redu ction assays gave similar portrayals of the kinetics of the macrophage resp iratory burst, while H(2)DCFDA did not. We therefore recommend the use of H 2DCF over H(2)DCFDA for quantification of the production of reactive oxygen species. Additionally, we stress the need to test reaction buffers or cult ure media used with H2DCF(DA) for their ability to oxidize the probe direct ly or indirectly. As an example, we have observed that tyrosine combined wi th ubiquitous metal contaminants of physiological buffers can result in hig h levels of oxidation, which may be incorrectly interpreted as cellular act ivity. (C) 2001 Elsevier Science B.V. All rights reserved.